The use of various membranes containing biologically active proteins, and especially membranes containing enzymes has become widespread in recent years. Also other means for the immobilization of enzymes have been resorted to.
In such enzyme electrodes the electrochemical sensors detect changes in the concentration of a product, a cosubstrate or a cofactor involved in the enzymatic conversion. The signal obtained is thus proportional to the concentration of the substrate. Enzyme electrodes are highly specific and allow for easy processing and control of repeated or continuous analysis (see Bowers, L. D. and Carr, P. W. Adv. Biochem. Eng. 15, 89-199, 1980).
In recent years several methods were developed for the construction of enzyme electrodes. These include physical entrapment of soluble enzyme by means of dialysis membrane (see Nilsson, H. et al. Biochem. Biophys. Acta 320, 529-534 (1973); physical gel entrapment of the enzyme in a gel layer, formed by radical polymerization of water soluble vinyl monomers in presence of the enzyme and fine nylon net (as a mechanical support) (see Nilsson, H. et al, ibid and Guilbault, G. G. and Nagy, G. Anal. Chem. 45, 417-419 (1973)); and crosslinking of the enzyme with inert protein by means of glutardialdehyde (see Durand, P. et al, BBA, 527, 277-281 (1978)).
The immobilization technique employed for the preparation of the enzyme membrane strongly affects the operational parameters of the enzyme electrode--the range of substrate concentrations that may be detected, response time, wash time and storage and operational stability.
The combination of enzyme membranes with electrochemical sensors, such as pH electrodes provides an efficient tool for use in analysis, research and fermentation industry.
One of the drawbacks of existing enzyme electrodes is the rather primitive attachment of the membrane in the pH electrode. Generally an attachment based on mechanical means, such as O-rings has been resorted to, generally with a protective material covering the membrane.
The present invention provides improved enzyme membranes and efficient means for the production of same. The method of production is fast, inexpensive and does not require complicated equipment. The novel enzyme electrodes have increased operational and storage capabilities compared with existing ones, increasing the efficiency and reproducibility of such devices.
The invention is illustrated in the following with reference to the production of enzyme electrodes. It ought to be clearly understood that the process of the invention can be used with a variety of other biologically active proteins, and that the resulting films or membranes can be bonded efficiently to various solid substrates due to their strong adhesivity to solids. They can be bonded to glass, porcelain, carbon and metals and thus various sensors and similar devices can be constructed.